Throughout this application various publications are referred to in parenthesis. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications are hereby incorporated by reference in their entireties into the subject application to more fully describe the art to which the subject application pertains.
The development of high-capacity microarrays has enabled the study of thousands of individual transcripts in parallel and helped identify the distinctive transcriptional profiles of tumors (Mohr et al. 2004). Consequently, the hierarchical clustering of tumor profiles has been demonstrated to be valuable for the classification of cancers. One such example has been the classification of gene expression patterns in primary breast tumors, which led to the identification of five distinct tumor subtypes subsequently linked with different clinical outcomes (Perou et al. 2000, Sorlie et al. 2001 and 2003). These studies substantiated the use of molecular taxonomy, in clinical medicine, for cancer diagnosis and identification of suited therapeutic approaches (Golub, 2001; Abramovitz and Leyland-Jones, 2006; Robison et al. 2004; Dietel et al. 2006).
Retrospective transcriptional profiling of archived tissues, which have been linked to long-term outcome of a disease, represent an attractive but challenging approach. The specimens that have been collected in surgical pathology have been routinely formalin-fixed and paraffin-embedded (FFPE), a preservation process that has been shown to induce the formation of cross-linkages between proteins and between proteins and nucleic acids (Werner et al. 2000). This fixation method has detrimental effects on RNA molecules that are only recovered fragmented, chemically modified and in low yields (Krafft et al. 1999; Stanta et al. 1998; Masuda et al. 1999; Coombs et al. 1999; Cronin et al. 2004). Multi-gene retrospective analyses of FFPE-RNA, which have been achieved through in-situ hybridization and relative quantification of target transcripts using real-time polymerase chain reaction (RT-PCR), have remained limited (Lehmann et al. 2001; Lewis et al. 2001; Relf et al. 2002; Capodieci et al. 2005; Paik et al. 2005). Although RT-PCR techniques have been enhanced for the study of larger gene sets, this technique remains impractical for the analysis of tens of thousands of genes and thus identification of early cancer related genes (Cronin et al 2004; Bibikova et al. 2004 a and b; Ma et al. 2006).
One major disadvantage to microarray analysis is the requirement of significant amounts of high-quality RNA, which are essential for increased sensitivity and reproducibility, a characteristic lacking with fragmented FFPE-RNA. Although a few commercial kits have been designed to reliably amplify small amounts of starting material, studies however have suggested that degraded FFPE-RNA is not a good substrate for cDNA synthesis prior to microarray experiments (Masuda et al. 1999; Karsten et al. 2002; Klur et al. 2004; Xiang et al. 2003; Wang et al. 2003). A few reports, describing the high-throughput transcriptional profiling of FFPE-RNA, have not provided any direct correlation with matched frozen tissues and therefore any indication on the percentage of gene retrieval (Onken et al. 2004; Chung et al. 2006).
Considering the high level of degradation of archived RNA, there is a need for a reliable assay for the preparation of highly fragmented and chemically modified FFPE-RNA, prior to in vitro transcription (IVT)-amplification and high-throughput analyses. This problem is solved by the strategy disclosed herein.